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Oncotarget-Poco Atf4 Resistencia
Oncotarget-Poco Atf4 Resistencia
Oncotarget-Poco Atf4 Resistencia
Research Paper
ABSTRACT
The uncontrolled growth of tumor can lead to the formation of area deprived in
nutrients. Due to their high genetic instability, tumor cells can adapt and develop
resistance to this pro-apoptotic environment. Among the resistance mechanisms,
those involved in the resistance to long-term amino acid restriction are not
elucidated. A long-term amino acid restriction is particularly deleterious since nine
of them cannot be synthetized by the cells. In order to determine how cancer cells
face a long-term amino acid deprivation, we developed a cell model selected for its
capacity to resist a long-term amino acid limitation. We exerted a selection pressure
on mouse embryonic fibroblast to isolate clones able to survive with low amino acid
concentration. The study of several clones revealed an alteration of the eiF2α/ATF4
pathway. Compared to the parental cells, the clones exhibited a decreased expression
of the transcription factor ATF4 and its target genes. Likewise, the knock-down of
ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover,
this association between a low level of ATF4 protein and the resistance to amino acid
deprivation was also observed in the cancer cell line BxPC-3. This resistance was
abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression
may be one important mechanism for cancer cells to survive under prolonged amino
acid deprivation.
INTRODUCTION lack of glucose and oxygen have been extensively studied [4,
5]. In contrast, less is known on how tumor cells can adapt to
Tumor cells are characterized by an uncontrolled the lack of lipids or amino acids. Since several years, there
proliferation rate. Such anarchic development of the tumor is an increasing interest for the role of the non-essential
mass may be accompanied by an inadequate vascularization amino acids (glutamine, serine, glycine,..) as precursors
of the tumors [1]. As a consequence, nutrients and oxygen of intermediate metabolites in the context of cancer [6, 7].
may not be provided sufficiently and could contribute to However, it is unclear how tumor cells can face a global
growth arrest and necrosis of the tumor [2]. However, tumor amino acid restriction since nine essential amino acids
cells exhibit high genetic instability which can lead to the cannot be synthesized by any mammalian cell. It remains
development of resistance mechanisms to nutrients and puzzling that some tumor cells facing such a deficit are able
oxygen deprived environment [3]. Mechanisms concerning not only to survive, but also to maintain minimal growth.
Figure 1: The AADR clones are resistant to the apoptosis induced by amino acid deprivation. (A) Proliferation assay of
parental cells (Parental) and 3 independent clones (Clone 1, 3, 5) cultured for 24, 48 and 120 hours in 2% medium (Concentration of amino
acid compared to the control medium). Cell numbers were determined by counting. For each cell type, cell number is expressed relative to
the number of cells at 0h. The graph show means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA;
*** indicates a significant difference (p<0,001) between clones and parental cells. (B) Early apoptosis detection using Hoechst 33342
labelling in parental cells and AADR clones cultured for 0, 24 and 48 hours in 2% medium. Results are expressed as the percentage of
cells presenting high Hoechst staining. Graph shows means ± S.E.M. of 3 independent experiments. Differences were assessed by 1-way
ANOVA; *** indicates a significant difference (p<0,001) between clones and parental cells. (C) Parental cells and AADR clones were
cultured in control medium (Ctl) or in 2% medium (2%) for 24h and 48h. Immunoblot analyses of caspase 3 cleavage were performed. (D)
Parental cells (Parental) and AADR clones (Clone 1, 2, 3) were cultured in medium lacking all amino acids for 48 hours and 72 hours. Cell
numbers were determined by counting. For each cell type, cell number is expressed relative to the number of cells at 0h. Graph shows means
± S.E.M. of 3 independent experiments. Differences were assessed by 1-way ANOVA; *** indicates a significant difference (p<0,001)
between parental cells and clones.
Figure 3: The expression of ATF4 protein and its target gene is diminished in the AADR clones. Parental cells (Parental)
and AADR clones (Clone 1, 2, 3) were cultured in control medium (Ctl) or in 2% medium (2%) for 24h and 48h. (A) (Left panel)
Immunoblot analyses of ATF4 and eIF2α were performed. (Right panel) ATF4 level was determined by densitometry analysis. Graph shows
means ± S.E.M. of 5 independent experiments. Differences were assessed by 1-way ANOVA. # indicates a significant difference (p<0,001)
compared to parental cells in control medium., ** indicates a significant difference (p<0,01) compared to parental cells in 2% medium. (B)
Parental cells and AADR clones were cultured in control medium or in 2% medium for 24h. Asns, Atf3, Atg12 and Trb3 mRNA levels were
determined and normalized by the level of β-actin mRNA, results are expressed relative to the value observed in parental cells in control
medium. Graph show means ± S.E.M. of 5 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant
difference (p<0,001) compared to parental cells in control medium, * p<0,05 compared to parental cells in 2% medium.
Figure 4: The knock-down of ATF4 expression in MEFs increases cell survival during amino acid deprivation. MEFs
were infected with a lentivirus expressing either a ShRNA control (ShCtl) or a ShRNA targeting ATF4 (ShATF4). (A) ShCtl cells (ShCtl)
and ShATF4 cells (ShATF4) were cultured in control medium (Ctl) or in 2% medium (2%) for 24 hours. (Left panel) Immunoblot analyses
of ATF4 and eIF2α were performed. (Right panel) ATF4 mRNA level was determined and normalized by the level of β-actin mRNA, results
are expressed relative to the value observed in ShCtl cells in control medium. Graph shows means ± S.E.M. of 3 independent experiments.
Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) compared to control medium *** indicates
a significant difference (p<0,001) compared to ShCtl cells in 2% medium. (B) ShCtl cells and ShATF4 cells were cultured in control
medium or in 2%medium for 24 and 48 hours. Immunoblot analyses of caspase 3 cleavage were performed. (C) ShCtl cells and ShATF4
cells were cultured in 2% medium for 5 days (D5) and 7 days (D7). Cell numbers were determined by counting. For each cell type, cell
number is expressed relative to the number of cells at 0h (represented by the black line). Graph shows means ± S.E.M. of 3 independent
experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,01) compared to ShCtl cells, **
indicates a significant difference (p<0,01) compared to 0h.
Figure 5: The expression of ATF4 is associated to the resistance to amino acid deprivation in the cancer cell line BxPC-
3. (A) HeLa, MIA PaCa-2 and BxPC-3 were cultured in 2% medium (2%) for 72 hours. Cell numbers were determined by counting. For
each cell type, cell number is expressed relative to the number of cells at 0h (represented by the black line). Graph shows means ± S.E.M. of
3 independent experiments. Differences were assessed by 1-way ANOVA; # indicates a significant difference (p<0,001) between cell lines,
** indicates a significant difference (p<0,01) compared to 0h. (B) MIA PaCa-2, BxPC-3 and HeLa cells were cultured control medium or
2% medium for 24 hours. Immunoblot analyses of ATF4, eIF2α and its phosphorylated form and the cleaved form of PARP were performed.
Figure 7: Summary diagram of the AADR clones. In the short term, the 2% amino acid medium induces rapidly the apoptosis of
mouse embryonic fibroblasts, through notably the induction of ATF4 expression. Nevertheless, this high selective pressure results in the
selection of Amino Acid Deprivation Resistant (AADR) clones. These clones present an alteration of the activation of the GCN2/eIF2α/
ATF4 pathway, despite the phosphorylation of GCN2 and eIF2α, the level of ATF4 remains low, preventing them from apoptosis.